Etanercept  formulations stabilized with xylitol

ABSTRACT

The invention provides stabilized aqueous pharmaceutical etanercept compositions suitable for long-term storage of etanercept, methods of manufacture of these compositions, methods of administration, and kits containing same.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation U.S. Ser. No. 15/716,005 filed Sep.26, 2017, which is a continuation of Ser. No. 15/209,484 filed Jul. 13,2016, now U.S. Pat. No. 9,770,510, which is a divisional of U.S. Ser.No. 13/654,919 filed Oct. 18, 2012, now U.S. Pat. No. 9,393,305, whichclaims the benefit of U.S. Provisional Application Nos. 61/669,480 filedJul. 9, 2012 and 61/548,518 filed Oct. 18, 2011, the disclosures ofwhich are incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to aqueous pharmaceutical compositionsstabilized with xylitol for long-term storage of etanercept, methods ofmanufacture of the compositions, methods of their administration, andkits containing the same. The invention includes etanercept formulationsthat do not require arginine for stabilization.

BACKGROUND OF THE INVENTION

Polypeptides must often be stored prior to their use. When stored forextended periods, polypeptides are frequently unstable in solution(Manning et al., 1989, Pharm. Res. 6:903-918). To extend their shelflife, additional processing steps have been developed, such as drying,e.g., lyophilization. However, lyophilized pharmaceutical compositionsare less convenient to use.

Typical practices to improve polypeptide stability can be addressed byvarying the concentration of elements with the formulation, or by addingexcipients to modify the formulation (See, for example, U.S. Pat. Nos.5,580,856 and 6,171,586). However, the use of additives can still resultin inactive polypeptides. In addition, in the case of lyophilization,the rehydration step can result in inactivation of the polypeptide by,for example, aggregation or denaturation (Nora et al., 1992, Pharm.Res., 9:33-36; Liu et al., 1991, Biotechnol. Bioeng., 37:177-184).Aggregation of polypeptides is undesirable, as it may result inimmunogenicity (Cleland et al., 1993, Crit. Rev. Therapeutic DrugCarrier Systems, 10:307-377; and Robbins et al., 1987, Diabetes,36:838-845).

Another way to improve polypeptide stability is to use L-arginine at aspecific concentration (U.S. Pat. No. 7,648,702).

One of the polypeptides that is stored for up to two years prior to useis etanercept (ENBREL® (etanercept), Immunex Corporation), which is adimeric fusion polypeptide consisting of the extracellularligand-binding portion of the human 75 kilodalton (p75) tumor necrosisfactor receptor (TNFR) linked to the Fc portion of human IgG1. Itconsists of 934 amino acids and has an apparent molecular weight ofapproximately 150 kilodaltons (Physicians Desk Reference, 2002, MedicalEconomics Company Inc.) The Fc component of etanercept contains theconstant heavy 2 (CH2) domain, the constant heavy 3 (CH3) domain andhinge region, but not the constant heavy 1 (CH1) domain of human IgG1.An Fc domain can contain one or all of the domains described above.Etanercept is usually produced by recombinant DNA technology in aChinese hamster ovary (CHO) mammalian cell expression system.

The present invention provides novel stable liquid formulations ofetanercept that allow its long-term storage.

SUMMARY OF THE INVENTION

The present invention is a stabilized aqueous pharmaceutical compositioncomprising etanercept and a stabilizer to inhibit instability,misfolding aggregation and/or fragmentation of the etanercept, whereinthe stabilizer comprises xylitol or a combination of xylitol andmeglumine.

Various technical terms used in the following discussion are definedbelow in the section entitled “Definitions” and throughout the remainderof the specification.

The stabilized etanercept formulations of the present invention whichare optionally and preferably free of arginine, elicit long term storagestability as characterized by at least one of (1) SEC analysis at M₃ orT₂ or T₄ of: monomer content greater than about 90%; aggregates contentof less than about 3 wt %; and fragment 3 content less than about 5 wt%: and (2) HIC analysis at M₃ or T₂, or T₄ wherein the amount of thecomposition represented by peak 1 of the HIC chromatogram is less thanabout 3 wt. %; the amount of the composition represented by peak 2 ofthe HIC chromatogram is greater than 80 wt. %; and the amount of thecomposition represented by peak 3 of the HIC chromatogram is less thanabout 20 wt %.

In preferred aspects of the stabilized formulations, the formulationselicit long term storage stability as characterized by: an HIC analysisat M₃ or T₂ or T₄ wherein the amount of the composition represented bypeak 2 of the HIC chromatogram is greater than or equal to about 95 wt.%; and wherein, if peak 3 is present on the HIC chromatogram, the amountof the composition represented by peak 3 is less than or equal to about3 wt. %.

The stabilized etanercept formulation as summarized above, optionallyand preferably, contains no arginine, or are essentially free ofarginine.

The formulations of the invention have excellent stability as determinedby SEC (Size Exclusion Chromatography) and HIC (Hydrophobic InteractionChromatography) analysis conducted after one, two or three months ofstorage at 5° C. These formulations are comparable to or better than acommercially available formulation of etanercept, in which arginine is arequired component. Accordingly the present invention is furtherdirected to formulations of stabilized etanercept, as summarized above,which contain no arginine, or are essentially free of arginine, andwherein the composition, evaluated at M₃ or T₂ or T₄, elicits long termstorage stability that meets one or both of the following criteria: (A)stability comparable to or better than commercially available etanerceptmarketed under the trademark ENBREL® (etanercept), as measured by (i)SEC analysis of the amounts of aggregate(s), monomer and fragment 3 inthe composition (as defined in the specification) and (ii) HIC analysisof amounts of material in the composition corresponding to peaks 1, 2and 3 of the HIC chromatogram (as defined in the specification); and (B)an HIC chromatogram in which (i) peak 3 is absent, or essentially absentand (ii) peak 2 represents greater than about 95 wt % of thecomposition; an SEC chromatogram containing essentially no peakcorresponding to aggregate(s); and an SEC chromatogram in which themonomer content represents at least about 95 wt % of the composition.

In one preferred aspect, the formulation of the invention comprisesabout about 25 to about 75 mg/ml of etanercept; and a stabilizer toinhibit instability, aggregation and/or fragmentation of the etanercept,wherein the stabilizer is xylitol in an amount constituting up to about10 wt. % of the composition, and wherein the composition ischaracterized by an SEC analysis at M₃ or T₂ or T₄ of greater than about80 wt. % monomer content; less than about 3 wt % aggregate(s) content;and less than about 6 wt % fragment 3 content and having a pH of 6.0 to6.6. Formulations meeting these analytical criteria do not requirearginine for stabilization.

In a further preferred embodiment the stabilized etanercept formulationis further characterized by (a) an SEC analysis at M₃ or T₂ or T₄ ofgreater than about 90 wt % monomer content; and less than about 3 wt %aggregate(s) content; and (b) an HIC analysis at M₃ or T₂ or T₄ whereinthe amount of the composition represented by peak 1 of the HICchromatogram is less than about 4 wt %; the amount of the compositionrepresented by peak 2 of the HIC chromatogram is greater than about 80wt %; and the amount of the composition represented by peak 3 of the HICchromatogram is less than about 20 wt %. Formulations meeting theseanalytical criteria do not require arginine for stabilization.

The etanercept compositions of the invention further afford the abilityto provide formulations which contain acceptable levels of subvisibleparticles. Accordingly, the invention is further directed to stabilizedetanercept formulations having, at M₃ or T₂ or T₄, no more than, onaverage, about 10,000 subvisible particles per mL having a size greaterthan 5 μm.

The stabilized etanercept composition of the present invention arefurther characterized by: (a) an SEC analysis at M₃ or T₂ or T₄ ofgreater than about 90 wt % monomer content; and less than about 3 wt %aggregate(s) content; and (b) an HIC analysis at M₃ or T₂ or T₄ whereinthe amount of the composition represented by peak 1 of the HICchromatogram is less than about 3 wt %; the amount of the compositionrepresented by peak 2 of the HIC chromatogram is greater than 80 wt %;and the amount of the composition represented by peak 3 of the HICchromatogram is less than about 20 wt % and wherein the formulation isfree or essentially free of arginine.

The stability of the formulations may be further characterized in thatthe compositions, optionally free or essentially of arginine, exhibit anHIC analysis at M₃ or T₂ or T₄ wherein the amount of the compositionrepresented by peak 1 of the HIC chromatogram is less than about 1%; theamount of the composition represented by peak 2 of the HIC chromatogramis greater than about 95 wt %; and the amount of the compositionrepresented by peak 3 of the HIC chromatogram is less than about 3 wt %.

Preferred stabilized compositions of the invention, preferably free oressentially free of arginine, exhibit an HIC analysis at M₃ or T₂ or T₄wherein the amount of the composition represented by peak 1 of the HICchromatogram is less than about 2% or preferably less than about 1%; theamount of the composition represented by peak 2 of the HIC chromatogramis greater than about 95 wt. % and preferably greater than about 97%;and the amount of the composition represented by peak 3 of the HICchromatogram is less than about 1 wt %, and preferably 0 to 1%.Formulations meeting these analytical criteria do not require argininefor stabilization.

The stabilized etanercept formulations may contain in addition toxylitol, the further optional ingredients meglumine, sucrose and sodiumchloride. Thus, the invention is further directed to compositionscomprising about 25 to 75 mg/ml of etanercept; about 1-10 wt % xylitol;about 1 to 30 mM sodium phosphate; optionally up to about 5 wt. %meglumine; optionally up to about 5 mM NaCl; and optionally up to about5 wt. % sucrose.

As differentiated from commercially available etanercept provided in anarginine-containing formulation, we found it surprising, in light ofU.S. Pat. No. 7,648,702, that the formulation embodiments of etanerceptdescribed and exemplified herein do not require arginine for long-termstabilization, although arginine may still be added if desired. Theability to provide etanercept formulations stabilized without argininerepresents a potentially significant benefit to the health care systemby providing patients and health care providers with alternativeformulations of etanercept that may become available at lower costcompared with present commercial etanercept formulation (i.e., Enbrel®)that require arginine for stabilization.

As used herein the term “instability” or like terms denotes the tendencyof the etanercept monomer to undergo a variety of undesiredtransformations during storage. Such transformations include theformation of oligomers and high molecular weight aggregate(s)(hereinafter terms “aggregate(s)” in which multiple copies of theessentially intact etanercept monomer become irreversibly associatedwith one another through a variety of non-covalent attractions (e.g.,electrostatic interactions.) Undesired transformations during storagemay also include degradation of the etanercept monomer to smallerfragments and/or dipped species. Ideally, a formulation of etanerceptshould minimize, to the greatest extent possible, the tendency of theformulation to result, during storage, in the formation of aggregates,misfolded protein, oligomers and/or fragments of etanercept. Animportant benefit resulting from the ability to reduce formation ofunwanted aggregates or fragments is a reduction in the potentialtoxicity and/or immunogenicity of the drug.

The etanercept formulation of the present invention which is optionallyand preferably free, or essentially free of arginine. The term“essentially free of arginine” is intended to mean that arginine, evenif present, is not contributing to the stabilization of the etanerceptmonomer in the formulation to such an extent that a person skilled inthe art would judge its presence beneficial or necessary from astabilization standpoint.

These and other aspects will become apparent from the followingdescription although variations and modifications therein may beaffected without departing from the spirit and scope of the novelconcepts of the disclosure.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the invention are now described in detail. Asused in the description and throughout the claims, the meaning of “a”,“an”, and “the” includes plural reference unless the context dearlydictates otherwise. Also, as used in the description and throughout theclaims, the meaning of “in” includes “in” and “on” unless the contextdearly dictates otherwise. Additionally, some terms used in thisspecification are more specifically defined below.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. Synonyms for certain termsare provided. A recital of one or more synonyms does not exclude the useof other synonyms. The use of examples anywhere in this specificationincluding examples of any terms discussed herein is illustrative only,and in no way limits the scope and meaning of the invention or of anyexemplified term. The invention is not limited to the variousembodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions will control.

“Around,” “about” or “approximately” shall generally mean within 20percent, within 10 percent, within 5, 4, 3, 2 or 1 percent of a givenvalue or range. Numerical quantities given are approximate, meaning thatthe term “around,” “about” or “approximately” can be inferred if notexpressly stated.

The term “etanercept” or “etanercept monomer” or “monomer” is synonymouswith Enbrel®. It refers to a polypeptide which is a dimeric fusionpolypeptide consisting of the extracellular ligand-binding portion ofthe human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR)linked to the Fc portion of human IgG1. It consists of 934 amino acidsand has an apparent molecular weight of approximately 150 kilodaltons.For the purposes of the present application, the term “etanercept” alsoencompasses etanercept with minor modifications in the amino acidstructure (including deletions, additions, and/or substitutions of aminoadds) which do not significantly affect the function, potency, oravidity of etanercept. The term “etanercept” encompasses all forms andformulations of Enbrel®, including but not limited to concentratedformulations, injectable ready-to-use formulations; formulationsreconstituted with water, alcohol, and/or other ingredients, and others.

The term “sugar” refers to monosaccharides, disaccharides, andpolysaccharides. Examples of sugars include, but are not limited to,sucrose, trehalose, dextrose, and others.

The term “meglumine” refers to a compound with chemical formulaH₃NHCH₂(CHOH)₄CH₂OH, also known as 1-Deoxy-1-methylaminosorbitol;N-Methyl-d-glucamine; and 1-Deoxy-1-methylamino-D-glucitol.

The terms “mannosylglycerate,” “mannosyllactate,” “mannosylglycolate,and “diglycerolphosphate” are well known in the art and have theircommonly accepted meanings. The following references describe thesecompounds in some detail: Faria et al., Carbohydrate Res. 2008, 343:3025-3033; Borges et al., Extremophiles 2002, 6: 209-216; Faria et al.,ChemBioChem 2003, 4: 734-741; Sawangwan et al., Biotechnol. J. 2010, 5:187-191; and Pals et al., J. Mol. Biol. 2009, 394: 237-250. Theapplication incorporates by reference the description of these compoundscontained in these references.

The term polyol” refers to an alcohol containing multiple hydroxylgroups. Examples of polyols include, but are not limited to, mannitol,sorbitol, and others.

The term “long-term storage” is understood to mean that thepharmaceutical composition can be stored for three months or more, forsix months or more, and preferably for one year or more. Long termstorage is also understood to mean that the pharmaceutical compositionis stored either as a liquid at 2-8° C., or is frozen, e.g., at −20° C.,or colder. It is also contemplated that the composition can be frozenand thawed more than once.

The term “stable” or “stabilized” with respect to long-term storage isunderstood to mean that etanercept contained in the pharmaceuticalcompositions does not lose more than 20%, or more preferably 15%, oreven more preferably 10%, and most preferably 5% of its activityrelative to activity of the composition at the beginning of storage.

The term “mammal” includes, but is not limited to, a human.

The term “pharmaceutically acceptable carrier” refers to a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material,formulation auxiliary, or excipient of any conventional type. Apharmaceutically acceptable carrier is non-toxic to recipients at thedosages and concentrations employed and is compatible with otheringredients of the formulation.

The term “composition” refers to a mixture that usually contains acarrier, such as a pharmaceutically acceptable carrier or excipient thatis conventional in the art and which is suitable for administration intoa subject for therapeutic, diagnostic, or prophylactic purposes. It mayinclude a cell culture in which the polypeptide or polynucleotide ispresent in the cells or in the culture medium. For example, compositionsfor oral administration can form solutions, suspensions, tablets, pills,capsules, sustained release formulations, oral rinses or powders.

The terms “pharmaceutical composition” and “formulation” are usedinterchangeably.

The term “treatment” refers to any administration or application ofremedies for disease in a mammal and includes inhibiting the disease,arresting its development, relieving the disease, for example, bycausing regression, or restoring or repairing a lost, missing, ordefective function; or stimulating an inefficient process. The termincludes obtaining a desired pharmacologic and/or physiologic effect,covering any treatment of a pathological condition or disorder in amammal. The effect may be prophylactic in terms of completely orpartially preventing a disorder or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disorder and/oradverse affect attributable to the disorder. It includes (1) preventingthe disorder from occurring or recurring in a subject who may bepredisposed to the disorder but is not yet symptomatic, (2) inhibitingthe disorder, such as arresting its development, (3) stopping orterminating the disorder or at least its associated symptoms, so thatthe host no longer suffers from the disorder or its symptoms, such ascausing regression of the disorder or its symptoms, for example, byrestoring or repairing a lost, missing or defective function, orstimulating an inefficient process, or (4) relieving, alleviating orameliorating the disorder, or symptoms associated therewith, whereameliorating is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, such as inflammation, pain and/or tumorsize.

The term “disease” refers to any condition, infection, disorder orsyndrome that requires medical intervention or for which medicalintervention is desirable. Such medical intervention can includetreatment, diagnosis and/or prevention.

The term “therapeutically effective amount” refers to an amount which,when administered to a living subject, achieves a desired effect on theliving subject. For example, an effective amount of the polypeptide ofthe invention for administration to the living subject is an amount thatprevents and/or treats an integrin αvβ3-mediated disease. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques. As isknown in the art, adjustments for systemic versus localized delivery,age, body weight, general health, sex, diet, time of administration,drug interaction and the severity of the condition may be necessary, andwill be ascertainable with routine experimentation by those skilled inthe art.

The term “T₁” refers to a point in time at which an etanerceptformulation has been stored for about one week at 40° C.

The term “T₂” refers to a point in time at which an etanerceptformulation has been stored for about two weeks at 40° C.

The term “T₄” refers to a point in time at which an etanerceptformulation has been stored for about four weeks at 40° C.

The term “M₃” refers, collectively, to three points in time, and inparticular to an analytical result being observed for an etanerceptformulation after duration of either about one, about two or about threemonths of storage at a storage temperature of 5° C. For example,reference herein to an analysis being conducted at M₃ should beunderstand to mean that such analysis is be done at the point in time atwhich etanercept formulation has been in storage for a duration selectedfrom about one, about two, or about three months. Thus, a requirementherein that an etanercept formulation elicit a certain analytical valueor measurement at M₃ is satisfied if the required value is observed at apoint in time corresponding to at least one of the following storagedurations: at approximately one month, at approximately two months or atapproximately three months of storage at 5° C.

The terms “Peak 1,” Peak 2” and “Peak 3” when used herein in connectionwith discussion of HIC chromatography results refers to the same peaks1, 2 and 3 discussed in U.S. Pat. No. 7,294,481.

EMBODIMENTS OF THE INVENTION

When pharmaceutical compositions containing etanercept (ENBREL®(etanercept)), including aqueous and lyophilized formulations ofetanercept are stored on a long term basis, the activity of etanerceptcan be lost or decreased due to instability of the etanercept monomervia aggregation and/or chemical degradation including formation offragments and oligomers. Thus, the present invention provides severalembodiments of aqueous formulations of etanercept that allow stablelong-term storage of etanercept, so that etanercept is stable over thecourse of storage either in liquid or frozen states. The providedformulations include, but are not limited to formulations that do notcontain arginine and do not require any extra steps such as rehydrating.

These embodiments are explained in a greater detail below.

Etanercept

All of the compositions of the present invention comprise etanercept(ENBREL®). As explained in the Background section of this application,etanercept is a dimeric fusion polypeptide consisting of theextracellular ligand-binding portion of the human 75 kilodalton (p75)tumor necrosis factor receptor (TNFR) linked to the Fc portion of humanIgG1. Etanercept consists of 934 amino adds. The Fc component ofetanercept contains the constant heavy 2 (CH2) domain, the constantheavy 3 (CH3) domain and hinge region of human IgG1. An Fc domain cancontain one or all of the domains described above.

Etanercept suitable for storage in the present pharmaceuticalcomposition can be produced by living host cells that expressetanercept, such as hybridomas in the case of antibodies, or host cellsthat that have been genetically engineered to produce the polypeptide inthe case of fusion polypeptides or antibodies. Methods of geneticallyengineering cells to produce polypeptides are well known in the art.See, e.g., Ausubel et al., eds. (1990), Current Protocols in MolecularBiology (Wiley, New York). Such methods include introducing nucleic addsthat encode and allow expression of the polypeptide into living hostcells. These host cells can be bacterial cells, fungal cells, or,preferably, animal cells grown in culture. Bacterial host cells include,but are not limited to, Escherichia coli cells. Examples of suitable E.coli strains include: HB101, DH5.alpha, GM2929, JM109, KW251, NM538,NM539, and any E. coli strain that fails to cleave foreign DNA. Fungalhost cells that can be used include, but are not limited to,Saccharomyces cerevisiae, Pichia pastoris and Aspergillus cells. A fewexamples of animal cell lines that can be used are CHO, VERO, BHK, HeLa,Cos, MDCK, 293, 3T3, and W138. New animal cell lines can be establishedusing methods well know by those skilled in the art (e.g., bytransformation, viral infection, and/or selection). Optionally,etanercept can be secreted by the host cells into the medium.

Purification of the expressed etanercept can be performed by anystandard method. When etanercept is produced intracellularty, theparticulate debris is removed, for example, by centrifugation orultrafiltration. When etanercept is secreted into the medium,supernatants from such expression systems can be first concentratedusing standard polypeptide concentration filters. Protease inhibitorscan also be added to inhibit proteolysis and antibiotics can be includedto prevent the growth of microorganisms.

Etanercept can be purified using, for example, hydroxyapatitechromatography, gel electrophoresis, dialysis, and affinitychromatography, and any combination of known or yet to be discoveredpurification techniques, including but not limited to Protein Achromatography, fractionation on an ion-exchange column, ethanolprecipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSET®, an anion or cation exchange resinchromatography (such as a polyaspartic add column), chromatofocusing,SDS-PAGE, and ammonium sulfate precipitation.

Etanercept Stabilized with Xylitol

The invention provides a stabilized aqueous pharmaceutical compositioncomprising etanercept and a stabilizer to inhibit instability,aggregation and/or fragmentation of the etanercept, wherein thestabilizer comprises xylitol or a combination of xylitol and meglumine.

Without wishing to be bound to any particular theory, it is believedthat xylitol reduces etanercept's tendency to associate in undesiredternary or quaternary complexes, and therefore, reduces aggregation ofetanercept. Presumably, xylitol acts as a conformational stabilizer,thereby reducing the propensity of etanercept to aggregate. Thereduction in aggregation is believed to last for a long period of time,e.g., two years or more. The stabilizing effects of xylitol are notlimited to reduction in aggregates but may involve other aspects ofstabilization of etanercept.

A preferred stabilized etanercept formulation incorporating xylitol forstabilization is one in which stabilization is provided by a combinationof xylitol and meglumine.

The pharmaceutical compositions of the invention may be prepared bycombining, a purified etanercept and xylitol, or xylitol in combinationwith meglumine. Further, a buffer, a tonicity modifier and an additionalexcipient and other commonly used inactive ingredients can be added asneeded. For simplicity, these are discussed more fully later in thespecification. A person of ordinary skill in the art will understandthat the combining of the various components to be included in thecomposition can be done in any appropriate order. For example, thebuffer can be added first, middle or last, and the tonicity modifier canalso be added first, middle or last. A person of ordinary skill in theart will also understand that some of these chemicals can beincompatible in certain combinations, and accordingly, are easilysubstituted with different chemicals that have similar properties butare compatible in the relevant mixture.

Xylitol stabilized etanercept formulations of the invention can compriseabout 25 to 75 mg/ml of etanercept; about 1-10 wt. % xylitol; about 1 to30 mM sodium phosphate; optionally up to about 5 wt. % meglumine;optionally up to about 5 mM NaCl; and optionally up to about 5 wt. %sucrose.

Xylitol stabilized etanercept formulations which additionally containmeglumine, sodium chloride and sucrose can comprise, in addition toxylitol, comprise about 1-100 mM NaCl; about 1 to 5 wt. % sucrose; andmeglumine in an amount of about 1-5 wt % of the composition.

In place of meglumine, the present invention also contemplates the useof mannosylglycerate, “mannosyllactate, mannosylglycolate, anddiglycerolphosphate.

In a further embodiment, xylitol stabilized etanercept formulations cancomprise about 25 to about 75 mg/ml of etanercept; and a stabilizer toinhibit instability, aggregation and/or fragmentation of the etanercept,wherein the stabilizer is xylitol in an amount constituting up to about10 wt. % of the composition, and wherein the composition ischaracterized by an SEC analysis at T₂ of: about 80 wt. % to about 95 wt% monomer content; an SEC analysis at T₂ of aggregate(s) content of lessthan about 4 wt. % and preferably less than about 3 wt %; and an SECanalysis at T₂ of fragment 3 content of less than about 8 wt. % andpreferably less than about 6 wt. %; wherein the composition has a pH ofabout 6.0 to about pH 7.0, and more preferably about 6.0 to about 6.6and most preferably about 6.3 to about 6.5.

In stabilized etanercept formulations such as those referenced abovecontaining xylitol or xylitol in combination with meglumine, theformulations are more preferably characterized by:

-   -   (a) an SEC analysis at T₄ of greater than about 90, 91, 92, 93,        94, 95, 96, or 97 wt. % monomer content; and less than about 3,        2 or 1 wt % aggregate(s) content; and    -   (b) an HIC analysis at T₂ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3, 2 or 1 wt. %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt. %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than about 20, 19, 18,        17, 16, 15, 14, or 13 wt %; and    -   (c) an HIC analysis at T₄ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3, 2 or 1 wt %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt. %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than 20, 19, 18, 17, 16,        15, 14 or 13 wt. %.

The terms “SEC”, “T₂” “T₄” “HIC” “monomer content” “aggregate(s)” and“fragment 3” “peak 1,” “peak 2,” and “peak 3,” are defined in theexamples below. Particularly preferred formulations containing xylitol,or xylitol in combination with meglumine are characterized by having anHIC analysis at T₄ or T₂ wherein the amount of the compositionrepresented by peak 1 of the HIC chromatogram is less than about 1%; theamount of the composition represented by peak 2 of the HIC chromatogramis greater than about 95 wt. % and preferably greater than about 99 wt.%; and the amount of the composition represented by peak 3 of the HICchromatogram is less than about 1 wt. %. Specific xylitol-stabilizedformulations are provided in the detailed examples.

Although the invention does not exclude the use of arginine, theetanercept formulations comprising xylitol for stabilization accordingto the present invention are free or essentially free of arginine.

Additional Components of the Provided Pharmaceutical Compositions

The formulations of the invention may also include buffers, tonicitymodifiers, excipients, pharmaceutically acceptable carriers and othercommonly used inactive ingredients of the pharmaceutical compositions.For simplicity, these are discussed more fully later in the application.

Buffers maintain pH in a desired range. Suitable buffers includehistidine, potassium phosphate, sodium or potassium citrate, maleicacid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris),various forms of acetate and diethanolamine. The concentration of thebuffer in the formulation is preferably between about 1 mM to about 1M,and more preferably about 10 mM to about 200 mM. Buffers are well knownin the art and are manufactured by known methods and available fromcommercial suppliers.

Examples of suitable buffers are phosphate, histidine, citrate, maleate,tartrate, succinate, acetate, tris-(hydroxymethyl)-aminomethane (tris),bicarbonate.

In a preferred embodiment, the buffer is sodium phosphate.

In a preferred embodiment, the pH of the pharmaceutical composition isat or near physiological levels. Thus, preferably, the pH of theprovided compositions is between about 5.8 and about 8.4; and even morepreferably, between about 6.2 and about 7.4. A person of ordinary skillin the art will understand that the pH can be adjusted as necessary tomaximize stability and solubility of etanercept in a particularformulation. Thus, etanercept formulations at a pH outside ofphysiological ranges, yet tolerable to the patient, are also within thescope of the invention.

A tonicity modifier is a molecule that contributes to the osmolality ofa solution. The osmolality of a pharmaceutical composition is preferablyadjusted to maximize the active ingredient's stability and/or tominimize discomfort to the patient upon administration. It is generallypreferred that a pharmaceutical composition be isotonic with serum,i.e., having the same or similar osmolality, which is achieved byaddition of a tonicity modifier.

In a preferred embodiment, the osmolality of the provided formulationsis from about 180 to about 420 mOsM. However, it is to be understoodthat the osmolality can be either higher or lower as specific conditionsrequire.

Examples of tonicity modifiers suitable for modifying osmolalityinclude, but are not limited to amino acids (not including arginine)(e.g., cysteine, histidine and glycine), salts (e.g., sodium chloride,potassium chloride and sodium citrate) and/or saccharides (e.g.,sucrose, glucose and mannitol).

Preferred tonicity modifiers are glycine, alanine, sodium chloride,potassium chloride, and sodium sulfate.

In a preferred embodiment, the concentration of the tonicity modifier inthe formulation is preferably between about 1 mM to about 1 M, morepreferably about 10 mM to about 200 mM. Tonicity modifiers are wellknown in the art and are manufactured by known methods and availablefrom commercial suppliers.

Excipients, also referred to as chemical additives, co-solutes, orco-solvents, that stabilize the polypeptide while in solution (also indried or frozen forms) can also be added to a pharmaceuticalcomposition. Excipients are well known in the art and are manufacturedby known methods and available from commercial suppliers.

Examples of suitable excipients include but are not limited tosugars/polyols such as: sucrose, lactose, glycerol, xylitol, sorbitol,mannitol, maltose, inositol, trehalose, glucose; polymers such as: serumalbumin (bovine serum albumin (BSA), human SA or recombinant HA),dextran, poly(viny alcohol) PVA, hydroxypropyl methylcellulose (HPMC),polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP),hydroxyethylcellulose (HEC); non-aqueous solvents such as: polyhydricalcohols, (e.g., PEG, and glycerol) and dimethylformamide (DMF); aminoacids such as: proline, L-serine, sodium glutamic acid, alanine,glycine, lysine hydrochloride, sarcosine and gamma-aminobutyric add;surfactants such as: Tween®-80 (polysorbate 80), Tween®-20 (polysorbate20), SDS, polysorbate, poloxamers; and miscellaneous excipients such as:potassium phosphate, sodium acetate, ammonium sulfate, magnesiumsulfate, sodium sulfate, trimethylamine N-oxide, betaine, metal ions(e.g., zinc, calcium, and magnesium), CHAPS, monolaurate,2-O-beta-mannoglycerate or any combination of the above.

Preferred excipients are sucrose, lactose, glycerol, xylitol, sorbitol,mannitol, maltose, inositol, trehalose, glucose, bovine serum albumin(BSA), human serum albumin (HSA), recombinant albumin, dextran, PVA,hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin,polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC), polyethyleneglycol, ethylene glycol, glycerol, alanine, glycine, lysinehydrochloride, sarcosine, SDS, polysorbate 20, polysorbate 80, poloxamer188, trimethylamine N-oxide, betaine, zinc ions, calcium ions, magnesiumions, CHAPS, sucrose monolaurate, and 2-O-beta-mannoglycerate.

The concentration of one or more excipients in a formulation of theinvention is/are preferably between about 0.001 to 5 weight percent,more preferably about 0.1 to 2 weight percent.

Methods of Treatment

In another embodiment, the invention provides a method of treating amammal comprising administering a therapeutically effective amount ofthe pharmaceutical compositions of the invention to a mammal, whereinthe mammal has a disease or disorder that can be beneficially treatedwith etanercept.

In a preferred embodiment, the etanercept is derived from the samespecies of mammal as is to be treated with the composition.

In a preferred embodiment, the mammal is a human.

Diseases or disorders that can be treated with the provided compositionsinclude but are not limited to rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Wegener's disease (granulomatosis),Crohn's disease (or inflammatory bowel disease), chronic obstructivepulmonary disease (COPD), Hepatitis C, endometriosis, asthma, cachexia,psoriasis, and atopic dermatitis. Additional diseases or disorders thatcan be treated with the compositions of the present invention includethose described in WO 00/62790, WO 01/62272, U.S. Patent Application No.2001/0021380, and U.S. Pat. No. 7,648,702 B2, the relevant portions ofwhich are incorporated herein by reference.

The provided pharmaceutical compositions may be administered to asubject in need of treatment by injection systemically, such as byintravenous injection; or by injection or application to the relevantsite, such as by direct injection, or direct application to the sitewhen the site is exposed in surgery; or by topical application.

In one embodiment, the invention provides a method of treatment and/orprevention of rheumatoid arthritis comprises administering to a mammalin need thereof a therapeutically effective amount of one of theprovided etanercept compositions.

The therapeutically effective amount of the etanercept in the providedcompositions will depend on the condition to be treated, the severity ofthe condition, prior therapy, and the patient's clinical history andresponse to the therapeutic agent. The proper dose can be adjustedaccording to the judgment of the attending physician such that it can beadministered to the patient one time or over a series ofadministrations.

In one embodiment, the effective etanercept amount per adult dose isfrom about 1-500 mg/m², or from about 1-200 mg/m², or from about 1-40mg/m² or about 5-25 mg/m².

Alternatively, a flat dose may be administered, whose amount may rangefrom 2-500 mg/dose, 2-100 mg/dose or from about 10-80 mg/dose.

If the dose is to be administered more than one time per week, anexemplary dose range is the same as the foregoing described dose rangesor lower and preferably administered two or more times per week at a perdose range of 25-100 mg/dose.

In another embodiment, an acceptable dose for administration byinjection contains 80-100 mg/dose, or alternatively, containing 80 mgper dose.

The dose can be administered weekly, biweekly, or separated by severalweeks (for example 2 to 8).

In one embodiment, etanercept is administered at 25 to 75 mg/ml by asingle subcutaneous (SC) injection.

In some instances, an improvement in a patient's condition will beobtained by administering a dose of up to about 100 mg of thepharmaceutical composition one to three times per week over a period ofat least three weeks. Treatment for longer periods may be necessary toinduce the desired degree of improvement. For incurable chronicconditions the regimen may be continued indefinitely. For pediatricpatients (ages 4-17), a suitable regimen may involve administering adose of 0.4 mg/kg to 5 mg/kg of etanercept, one or more times per week.

In another embodiment, the pharmaceutical formulations of the inventionmay be prepared in a bulk formulation, and as such, the components ofthe pharmaceutical composition are adjusted to be higher than would berequired for administration and diluted appropriately prior toadministration.

The pharmaceutical compositions can be administered as a soletherapeutic or in combination with additional therapies as needed. Thus,in one embodiment, the provided methods of treatment and/or preventionare used in combination with administering a therapeutically effectiveamount of another active agent. The other active agent may beadministered before, during, or after administering the pharmaceuticalcompositions of the present invention. Another active agent may beadministered either as a part of the provided compositions, oralternatively, as a separate formulation.

Administration of the provided pharmaceutical compositions can beachieved in various ways, including parenteral, peroral, buccal,sublingual, nasal, rectal, intraperitoneal, intradermal, transdermal,subcutaneous, intravenous, intra-arterial, intracardiac,intraventricular, intracranial, intratracheal, intrathecaladministration, intramuscular injection, intravitreal injection, andtopical application.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, i.e., subcutaneously,intramuscularly, intravenously, intraperitoneal, intracerebrospinal,intra-articular, intrasynovial, intravitreal, and/or intrathecal.Parenteral administration can be by bolus injection or continuousinfusion. Pharmaceutical compositions for injection may be presented inunit dosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. In addition, a number of recent drug deliveryapproaches have been developed and the pharmaceutical compositions ofthe present invention are suitable for administration using these newmethods, e.g., Inject-ease®, Genject, injector pens such as GenPen®, andneedleless devices such as MediJector® and BioJector®. The presentpharmaceutical composition can also be adapted for yet to be discoveredadministration methods. See also Langer, 1990, Science, 249:1527-1533.

The provided pharmaceutical compositions can also be formulated as adepot preparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the formulations may bemodified with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions may, if desired, be presented in a vial,pack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. In one embodiment the dispenser devicecan comprise a syringe having a single dose of the liquid formulationready for injection. The syringe can be accompanied by instructions foradministration.

In another embodiment, the present invention is directed to a kit orcontainer, which contains an aqueous pharmaceutical composition of theinvention. The concentration of the polypeptide in the aqueouspharmaceutical composition can vary over a wide range, but is generallywithin the range of from about 0.05 to about 20,000 micrograms permilliliter (μg/ml) of aqueous formulation. The kit can also beaccompanied by instructions for use.

The present invention is more particularly described in the followingexamples that are intended as illustrative only, since manymodifications and variations therein will be apparent to those skilledin the art.

Example Etanercept Stabilized with Xylitol

Etanercept formulations stabilized with xylitol, preferably withoutarginine, may be prepared and tested using the procedures generallydescribed below.

Each solid formulation component is weighed to the amount required for agiven volume of formulation buffer. These components are combined into abeaker or vessel capable of carrying and measuring the given volume offormulation buffer. A volume of deionized water equal to approximately ¾of the target given formulation buffer is added to the beaker, and thecomponents are then solublized. The pH of the buffer is adjusted to thetarget formulation pH using 1 M sodium hydroxide and/or 1 M hydrogenchloride. The final formulation buffer volume is then raised to thetarget volume through the addition of deionized water. Etanerceptprotein solution is placed in dialysis material housing (such as ThermoScientific Slide-A-Lyzer MINI Dialysis Unit 10,000 MWCO), which is thenplaced in contact with the desired formulation buffer for 12 hours at 4°C. Formulation buffer volume to protein solution volume ratio should beno less than 1000:1. The dialysis housing and protein solution itcontains is then placed in a second, equal volume of formulation bufferfor an additional 12 hours at 4° C. Resulting protein solution isremoved from the dialysis material housing, and the concentration ofprotein determined using ultraviolet spectroscopy. Protein concentrationis adjusted to the desired level using centrifugation (such as AmiconUltra 10,000 MWCO Centrifugal Concentrators) and/or dilution withformulation buffer.

The compositions can be tested for long-term stability by size exclusionchromatography (SEC), denatured SEC (dSEC), hydrophobic interactionchromatography (HIC), sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE), and for binding and bioactivity at varioustimepoints. The bioactivity can be measured by any number of well-knownassays.

For example, the techniques of Size Exclusion Chromatography aredescribed in Hawe at al, Pharm. Res. 2011, 28: 2302 and/or vanMarrschalkerweerd et al., Eur. J. Pharm. Biopharm. 2011, 78: 213.Similarly, the techniques of Denatured Size Exclusion Chromatography,Hydrophobic Interaction Chromatography, and SodiumDodecylSulfate-PolyAcrylamide Gel Electrophoresis are also well known topersons having ordinary skill in the art.

It is believed that the composition will be stable over the term of twoyears or more. The compositions exemplified below do not containarginine.

Formulation 1:17

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.3 (inactive) 25 mM Xylitol (inactive) 10% (w/v)

Formulation 2:10

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.31 (inactive) 25 mM Xylitol (inactive) 6% (w/v)

Formulation 2:11

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.3 (inactive) 25 mM Xylitol (inactive) 2.5% (w/v) Sucrose(inactive ingredient) 5% (w/v)

Formulation 2:18

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.4 (inactive) 25 mM Xylitol (inactive) 2.5% (w/v)Meglumine (inactive) 2.5% (w/v)

Formulation 2:19

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Sodiumphosphate, pH 6.24 (inactive) 10 mM Xylitol (inactive) 2.5% (w/v)Meglumine (inactive) 2.5% (w/v) NaCl (inactive) 2.5% (w/v) Sucrose(inactive) 1% (w/v)

Example 2 Preparation of Etanercept

Step 1. Cell Expansion.

In a manner known in the art, cell expansion necessary to generate asufficient number of cells for inoculation of a production bioreactor isperformed using a clone of CHO cells expressing the etanercept fusionprotein. The product of this expression process (a harvested cellculture fluid) results in a mixture of correctly folded etanercept, aswell as incorrectly folded and/or aggregated etanercept, along withadditional impurities. The harvested cell culture fluid comprising suchprotein mixture is subjected to detergent viral inactivation.

Step 2.

Affinity Chromatography. Affinity chromatography is performed on theharvested cell culture obtain in Step 1 above using a conventionalProtein A affinity column in a well known manner. Product recovery isapproximately 85%. The product obtained is a complex protein mixturecomprising correctly folded etanercept, incorrectly folded etanercept,and/or aggregates of correctly and/or incorrectly folded etanercept, orprotein fragments. The product obtained from this Protein A affinitycolumn purification step is adjusted to pH 3.5 and then subjected to aviral inactivation step. Following viral inactivation the product isadjusted to pH 5.5 and then further clarified in a known manner using acommercially obtained capsule filter.

Step 3A.

Mixed-Mode Cation Exchange Chromatography. A 31.8 L (45 cm diameter×20cm bed height) packed bed GE Healthcare Capto MMC chromatography columnis used to purify the product obtained in Step 2 above. Prior to use,the column is equilibrated with 2 CV of 25 mM acetate pH 5.5 andsanitized with 2 CV of 0.1 N NaOH, 1 M NaCl and neutralized with 2 CV of25 mM acetate, 0.7 M NaCl, pH 5.5. The column is then equilibrated with8-10 CV of 25 mM acetate pH 5.5 until the effluent is pH 5.5 and 3.5mS/cm. The Protein A pool from step 2 above is diluted to ≤6 mS/cm withWFI and applied to a column loading of up to 15 g/L media for eachcycle. The column is operated at a linear velocity of 200 cm/h to give a6 minute residence time. After loading, the column is washed with 2 CVof 25 mM acetate pH 5.5. The product is then eluted with an 8.5 CV, 15%to 85% gradient of 25 mM acetate pH 5.5 to 25 mM acetate, 0.7 M NaCl, pH5.5. Product collection begins at 0.15 OD (A280, 1.0 cm path length) andcollection ends at 50% of peak maximum. The eluate volume isapproximately 5 CV. Residual product and contaminants are stripped fromthe column with 2 CV of 10 mM Tris, 1 M NaCl, pH 8.0 and discarded. Theproduct obtained from the mixed mode column is filtered using aMillipore Opticap XL10, 0.22 μm Durapore capsule filter, (0.69 m²). Theproduct obtained from this step represents a recovery of about 70% ofthe Protein A material obtained in Step 2

Step 3B.

Mixed Mode Anion Exchange Chromatopgraphy. A 27.0 L (45 cm diameter×17cm bed height) packed bed GE Healthcare Capto Adhere chromatographycolumn is used to further purify the product obtained in step 3A above.Prior to use, the column is equilibrated with 2 CV of 25 mM Tris, pH 8.0and sanitized with 2 CV of 0.1 N NaOH, 1 M NaCl and neutralized andequilibrated with 2 CV of 25 mM Tris, pH 8.0. Prior to product loading,the column is equilibrated with 3 CV of 10 mM Tris, pH 8.0. The CaptoMMC pool from Step 3A above is adjusted to pH 8.1 with ˜0.045 kg of 1 MTris, pH 8.3 per kg of pool. The product from Step 3A above was dilutedin-line 1:3.8 with WFI to adjust the conductivity to 12.0 mS/cm and pH8.0. The resulting material is then applied to a column loading of up to15 g/L media. The column is operated at a linear velocity of 170 cm/h togive a 6 minute residence time. After loading, the column is washed with2 CV of 25 mM Tris, pH 8.0. The product is then eluted with a 10 CVgradient (20% to 90%) of 25 mM Tris, pH 8.0 to 10 mM Tris, 1 M NaCl, pH8.0. Product collection is started at 0.15 OD (A280, 1.0 cm path length)and collection ended at 25% of peak maximum. The eluate volume is 4-6CV. The eluted product is filtered using a commercially availablecapsule filter and then subjected in a known manner to viral filtrationand tangential flow filtration steps. Overall product recovery from step3B (including the final viral and tangential flow filtration steps) wasapproximately 68%. Product recovery measured before the filtration stepswas about 75%. A schematic representation of HIC data obtained oneluation fractions from this step are representing in FIG. 12.

Analysis:

The final filtered product obtained in this example is found to havegreater than about 90 wt % correctly folded etanercept as determined byHIC; less than 5 wt % incorrectly folded etanercept species asdetermined by HIC; less than about 3 wt % of dipped material by HICanalysis (believed to be fragments of etanercept in which the TNFRportion thereof has been truncated) and a combined amount of correctlyand incorrectly folded etanercept of greater than 95 wt % as determinedby size exclusion chromatography.

Analysis of Etanercept Formulations

A. Thermal Stability Storage

Following dialysis and concentration, samples of the etanerceptformulations exemplified above were sterile filtered in a bio safetycabinet. Using sterilized pipettes and autoclaved pipette tips, samplesof the etanercept formulations were transferred to pre-labeled andautoclaved 1 mL lyophilization vials. Vials were stoppered with sterilebutyl stoppers and crimped with aluminum caps. All vials were thentransferred to thermal stability ovens. Samples were subject to twothermal stability regimes: (1) two weeks at 40° C. and (2) four weeks at25° C. Throughout this specification, these two temperature regimes aredenoted “T₁” and T₄,” respectively.

B. Size Exclusion Chromatography (SEC)

Etanercept formulations disclosed herein were analyzed using the wellknown technique of Size Exclusion Chromatography (SEC), ahigh-performance liquid chromatography method in which analytes areseparated by size (see Rogner, M. (2000). Size Exclusion Chromatography.Protein Liquid Chromatography. M. Kastner. Amsterdam, Elsevier. 681:89-145.). In order to evaluate thermal stability of the Etanerceptsamples described above, the samples were examined by a SEC method basedon the literature (van Maarschalkerweerd, A., G. J. Wolbink, et al.(2011). “Comparison of analytical methods to detect instability ofetanercept during thermal stress testing.” European Journal ofPharmaceutics and Biopharmaceutics 78(2): 213-221.) The mobile phasebuffer was prepared to contain 50 mM sodium phosphate monobasicmonohydrate and 150 mM arginine. The pH was adjusted to 6.5 using 1 MHCl. All separations were performed using a Tosoh TSK-Gel SWxl 6 mm×4 cmguard column (cat no. 8543) attached linearly to a Tosoh TSK-Gel G4000SWxl 7.8 mm×30 cm (cat. no. 8542). To perform a separation, the columnswere brought to room temperature (23° C.) and equilibrated with mobilephase at a flow rate of 0.5 mL/min. 5 microliters of 50 mg/mL etanerceptformulation were injected onto the column using an autosampler. Theseparation was accomplished over 30 minutes at a flow rate of 0.5mL/minute. Column eluent was monitored at a wavelength of 280 nm duringthis time.

C. Integration of Size Exclusion Chromatography Chromatograms

All integration was performed using Chromeleon software (Dionex). Priorto integration, the SEC chromatogram for a buffer containing noetanercept was subtracted from all chromatograms. All integration wasperformed between retention times of 12 minutes and 26 minutes. Severalparameters were used to define a peak. The minimum area for a detectedpeak was set to 0.05 mAu*min. The two-dimensional sensitivity for peakdetection was set to 0.01 mAu and 75 seconds. Peak shoulders were addedmanually using a manual integration tool. All detected peaks weremanually adjusted in two steps. First, peak baselines (the bottomboundary of the peak) were adjusted to horizontal. Secondly, thevertical positions of the peak baselines were adjusted to that of thechromatogram baseline. The chromatogram baseline value was defined asthe signal in absence of analyte. The signal in absence of analyte wasdefined as the absorbance in mAu at 12 minutes retention time.

D. SEC Fractions of Etanercept Formulations

In the SEC analysis of etanercept formulations described above, threeSEC chromatography fractions were identified and studied. The fractionsthat were analyzed were, in the order of elution from the SEC column:(1) a high molecular weight fraction representing aggregates of theintact etanercept TNFR:FC fusion protein likely assembled vianon-covalent electrostatic attraction among intact etanercept molecules(hereinafter “aggregate(s)” or aggregate(s) content); (2) monomercontent, representing the intact etanercept TNFR:Fc fusion protein(hereinafter referred to as “monomer” of “monomer content”); (3) afraction likely representing one fragment or a population of fragmentsof the etanercept molecule in which one portion of the TNFR:moleculefusion protein has become cleaved from the monomer; in the loss of anarm of the Fab portion of the fusion protein at the hinge region of themolecule. The most common fragment or dipped species, as measured bySEC, is referred to as fragment 3. In conducting the SEC analysis, itwill be observed that aggregates elute first, followed by monomer,followed by fragment 3.

The following tables shows the relative amounts of Aggregates, Monomerand fragment 3 determined by SEC analysis as described above.

TABLE 1 SEC ANALYSIS OF MONOMER Formulation No. t₀ t₁ t₂ CommercialEnbrel 98.81 92.58 87.64 (comparative) [1:2] 1:17 98.02 93.90 87.53 2:1098.09 — 87.56 2:11 98.10 — 88.03 2:18 98.10 — 89.19 2:19 98.19 — 89.63Note: Amounts reported Tables I, II and III are percentages by weight T₀= formulation maintained at 5 C. and analyzed within 24 hours ofcreation. T₁ = formulation stored for one week at 40° C. T₂ =formulation stored for two weeks at 40 C.

TABLE II SEC ANALYSIS OF AGGREGATES Formulation No. t₀ t₁ t₂ CommercialEnbrel 0.09 0.59 1.02 (comparative) 1:17 0.31 0.70 2.17 2:10 0.29 — 2.572:11 0.31 — 1.68 2:18 0.29 — 1.53 2:19 0.26 — 1.24

TABLE III ANALYSIS OF FRAGMENT 3 Formulation No t₀ t₁ t₂ CommercialEnbrel 0.00 3.30 6.29 (comparative) 1:17 0.00 2.33 4.10 2:10 0.00 — 5.102:11 0.00 — 5.68 2:18 0.00 4.24 2:19 0.00 4.34

HIC Analysis of Etanercept Formulations

HIC chromatography may be carried out in a manner known in the art andgenerally described in U.S. Pat. No. 7,294,481, incorporated herein byreference. Samples are evaluated at to (within 24 hours of preparationat 5° C.) and again after either two weeks of storage at 25° C. (t₂) orafter 4 weeks of storage at 25° C. In the HIC chromatograms of theformulations of the present invention, Peak 1 in the HIC chromatogram isbelieved to be or include “Fragment 3”, which is identified andquantified using SEC, as referenced above in the discussion of SEC data;Peak 2 is etanercept monomer as referenced above in the discussion ofSEC data; and Peak 3 includes “Aggregate(s)” as referenced above in thediscussion of SEC data. It should further be understood that the terms“peak 1”, “peak 2” and “peak 3 as used here also constitute a referenceto the HIC peak 1, peak 2 and peak 3 referred to and disclosed in FIG. 4of U.S. Pat. No. 7,294,481 incorporated herein by reference.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1. A method of reducing formation of etanercept aggregates or fragmentsin an aqueous composition containing about 50 mg/ml etanercept, themethod comprising combining a) etanercept, b) buffer, and c) stabilizercomprising 6 to 10 weight % xylitol, wherein the aqueous composition hasa pH of 6.0 to 6.6, or a pH within 10 percent of 6.0 and 6.6, whereinthe aqueous composition is free or essentially free of arginine, therebypreparing a stable aqueous etanercept composition.
 2. The method ofclaim 1, wherein the stable aqueous etanercept composition comprises 10wt. % xylitol.
 3. The method of claim 1, wherein the stable aqueousetanercept composition comprises 25 to 75 mg/mL of etanercept.
 4. Themethod of claim 1, wherein the stable aqueous etanercept compositioncomprises 50 mg/mL or a concentration within 10 percent of 50 mg/ml ofetanercept.
 5. The method of claim 1, further comprising adding one ormore additional components selected from a buffer, a tonicity modifier,and an excipient to the aqueous pharmaceutical composition.
 6. Themethod of claim 1, further comprising adding one or more additionalcomponents selected from meglumine, mannosylglycerate, mannosyllactate,mannosylglycolate, and diglycerolphosphate to the aqueous pharmaceuticalcomposition.
 7. The method of claim 1, wherein the stable aqueousetanercept composition has osmolality of 180 to 420 milliosmoles or anosmolality within 10 percent of 180 to 420 milliosmoles.
 8. The methodof claim 1, further comprising filling the stable aqueous etanerceptcomposition in a device suitable for administration to a subject as asingle dose.
 9. The method of claim 8, wherein the dose contains 10 to100 mg/dose etanercept.
 10. The method of claim 1, wherein the stableaqueous etanercept composition elicits long term storage stability ascharacterized by SEC (size exclusion chromatography) analysis at T₂ ofmonomer content greater than 90%.
 11. The method of claim 1, wherein thestable aqueous etanercept composition has no more than, on average,10,000 subvisible particles per mL having a size greater than 5 μm. 12.The method of claim 1, wherein the stable aqueous etanercept compositionelicits long term storage stability as characterized by at least one of:(a) SEC (Size Exclusion Chromatography) analysis at T₂ of: monomercontent greater than 80 or 90%; aggregates content of less than 3 wt. %;and fragment 3 content less than 5 or 6 wt. %; or (b) HIC (HydrophobicInteraction Chromatography) analysis at T2 wherein the amount of thecomposition represented by peak 1 of the HIC chromatogram is less than 3wt. %; the amount of the composition represented by peak 2 of the HICchromatogram is greater than 80 wt. %; and the amount of the compositionrepresented by peak 3 of the HIC chromatogram is less than 20 wt. %. 13.The method of claim 12, wherein the stable aqueous etanerceptcomposition has an HIC analysis at T₂ wherein the amount of thecomposition represented by peak 1 of the HIC chromatogram is less than 1wt. %; the amount of the composition represented by peak 2 of the HICchromatogram is greater than 95 wt. %; and the amount of the compositionrepresented by peak 3 of the HIC chromatogram is less than 3 wt. %. 14.The method of claim 1, wherein the stable aqueous etanercept compositionelicits long term storage stability as characterized by an SEC (SizeExclusion Chromatography) analysis at T₂ of: greater than 80 wt. %monomer content; less than 3 wt. % aggregate(s) content; and less than 6wt. % fragment 3 content.
 15. A method of treating a subject in need oftreatment by injection of etanercept comprising administering to thesubject a stable aqueous etanercept composition comprising 25 to 75mg/ml etanercept and 6 to 10 wt. % xylitol in an aqueous pharmaceuticalcomposition having a pH of about 6.0 to 6.6 or a pH within 10 percent of6.0 and 6.6, wherein the composition is free or essentially free ofarginine.
 16. The method of claim 15, comprising administering 10 to 100mg etanercept per dose to the subject.
 17. The method of claim 15,comprising injecting the stable aqueous etanercept compositionsubcutaneously or intramuscularly.
 18. A method of treating a subject inneed of treatment for rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, Wegener's disease (granulomatosis), Crohn'sdisease (or inflammatory bowel disease), chronic obstructive pulmonarydisease (COPD), Hepatitis C, endometriosis, asthma, cachexia, psoriasis,or atopic dermatitis comprising administering to the subject a stableaqueous etanercept composition comprising 25 to 75 mg/ml etanercept and6 to 10 wt. % xylitol in an aqueous pharmaceutical composition having apH of about 6.0 to 6.6 or a pH within 10 percent of 6.0 and 6.6, whereinthe composition is free or essentially free of arginine.
 19. The methodof claim 18, comprising administering 10 to 100 mg etanercept per doseto the subject.
 20. The method of claim 18, wherein the administeringcomprises injecting the stable aqueous etanercept compositionsubcutaneously or intramuscularly.
 21. A vial, syringe, or injector pencontaining a stable aqueous etanercept composition comprising about 25to 75 mg/ml etanercept and 6 to 10 wt. % xylitol in an aqueouspharmaceutical composition having a pH of about 6.0 to 6.6 or a pHwithin 10 percent of 6.0 and 6.6, wherein the composition is free oressentially free of arginine.